It is now well established that the parameters of GR-mediated gene induction (Amax, EC50, and PAA) can be modulated by changing the concentrations of involved transcriptional cofactors. Furthermore, we have recently reported that the different parameters can be controlled by different domains of the modulatory proteins. Thus, it is possible that only one or two, as opposed to all three, parameters may change under certain conditions (Awasthi and Simons, 2012, Mol Cell Endocrinol, 355, 121-134). Our earlier studies in human peripheral mononuclear cells (PBMCs) have confirmed that such changes with varying factor concentration affect the induction parameters of endogenous, as well as exogenous, GR-regulated genes (Luo and Simons Jr., 2009, Human Immunology, 70, 785-789). These results provide strong support for our hypothesis that the modulation of GR induction parameters is a relevant feature of human physiology. Current conventional assays determine the temporal ordering of cofactor association with DNA. However, almost all of the available mechanistic conclusions are phenomenological. In our continuing collaboration with Carson Chow, we have extended our experimentally supported mathematical theory of steroid hormone action to describe antisteroid action and the changes in PAA while using glucocorticoid receptors as a model system. The theory shows why changes in PAA cannot be explained simply by differences in ligand affinity for receptor and requires action at a second step or site in the overall sequence of reactions. The theory also provides a method for locating the position of this second site, relative to a concentration limited step (CLS), which is a previously identified step in glucocorticoid-regulated transactivation that always occurs at the same position in the overall sequence of events of gene induction. Finally, the theory predicts that classes of antagonist ligands may be grouped on the basis of their maximal PAA with excess added cofactor and that the members of each class differ by how they act at the same step in the overall gene induction process. Thus, this theory now makes it possible to predict how different cofactors modulate antisteroid PAA, which should be invaluable in developing more selective antagonists. We have continued to use our theoretical model to uncover new cofactors affecting glucocorticoid regulated gene transcription Zhang et al., 2013, J Biol Chem, 288, 42-58; Zhu et al., 2014, Biochemistry, 53, 1753-1767. In collaboration with Dinah Singer (NCI), we find that BRD4 alters both the Amax and the EC50 of GR-mediated gene expression by acting at a minimum of three different kinetically-defined steps. The action at two of these steps are concentration dependent and, in one case, contingent on BRD4s interaction with P-TEFb. BRD4 is also shown to bind to NELF-E, a component of the NELF-complex, but to function before the site of NELF-E action. Importantly, the ability of NELF-E to modify GR induction is independent of the NELF complex. Several of the kinetically-defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR controlled gene induction have also been uncovered by the competition assay. In summary, we are applying both novel and conventional methodologies to obtain previously unavailable molecular information not only about the determinants of glucocorticoid steroid activity but also about the modulation of the total activity (Amax) and dose-response curve (EC50) of agonists plus the partial agonist activity (PAA) of antisteroids. The latter information stems from both a theoretical understanding of antisteroid action and a rational approach to identify cofactors acting at specific steps in steroid hormone action. These modulatory factors permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. More importantly, the identification of factors influencing steroid hormone action at sites well downstream of steroid binding, and close to the final, desired effect, offer the prospect of modifiable targets that should evoke fewer undesirable side effects due to their proximity to the final response. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.